謝秀梅Hsieh, Hsiu-Mei柯雅云Ko, Ya-Yun2019-09-052024-12-312019-09-052019http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22G060543027S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/103946阿茲海默氏症為一種最常見的失智症，分為兩種類型：早發性的家族遺傳型阿茲海默氏症(Familial Alzheimer’s disease, FAD)及晚發性的偶發型阿茲海默氏症(Sporadic Alzheimer’s disease, SAD)。家族遺傳型阿茲海默氏症是因為基因的突變所導致，如：類澱粉蛋白前驅蛋白(amyloid precursor protein, APP)、早老素(Presenilin-1, PS1、Presenilin-2, PS2)的突變；而偶發型阿茲海默氏症可能受環境、飲食習慣、壓力及年紀等因素影響。阿茲海默氏症主要的病徵有β類澱粉蛋白(Aβ)堆積在細胞外形成類澱粉斑塊，及tau蛋白過度磷酸化導致神經細胞內的神經纖維糾結，而這些最終都會導致神經細胞的死亡。在過去幾十年，臨床的實驗只能夠延緩阿茲海默氏症病程的發展，卻無法有效的治療這個疾病。良好的動物模式對於實驗的進行是十分重要的，但在實驗室以往的經驗中我們發現B6.129-Psen1tm1Mpm Tg (APPSwe, tauP301L) 1Lfa/J這種3基因轉殖鼠所顯現的性狀並不明顯，而且不容易準備相同遺傳背景的野生鼠作為實驗控制組。為了要有一個更具代表性的實驗動物，我們將此基因轉殖鼠的品系從B6.129雜交品系回交配成B6的純品系，並以行為實驗進行B6純品系3基因轉殖小鼠短期記憶、長期記憶、焦慮、運動能力的評估。我們發現B6.129雜交3基因轉殖小鼠不論在6、9、12、18月大，運動能力都顯著低於野生型B6小鼠及B6純品系 3基因轉殖小鼠，而B6純品系 3基因轉殖小鼠與野生型B6小鼠之間沒有顯著的差異，但在焦慮行為測試中顯示B6.129雜交鼠較無焦慮情形。另外利用西方墨點法測量小鼠海馬迴各種蛋白質的表現量，我們發現12月大B6.129雜交3基因轉殖小鼠及18月大B6純品系3基因轉殖小鼠則在Aβ的表現量相較於野生型小鼠有顯著的上升。Tau蛋白的表現量及202、396位點磷酸化程度則是兩種品系的3基因轉殖小鼠從6月大起都顯著的高於野生型小鼠，而腦片染色結果也可以發現B6純品系3基因轉殖小鼠及B6.129雜交3基因轉殖小鼠從9個月大開始其Tau蛋白202位點的螢光強度就顯著的高於野生型小鼠。除此之外，在檢查多種磷酸化Tau蛋白的激酶後我們發現12及18月大B6品系的3基因轉殖小鼠pERK的表現量顯著的高於野生型及B6.129雜交3基因轉殖小鼠，我們發現pERK的訊號與NeuN的訊號有共表現的狀況，確認ERK的活化是在神經細胞中，因此我們認為Tau蛋白的磷酸化可能是經由ERK活化所引起的。而利用腦片染色方式檢測小鼠腦中Iba1及GFAP數量發現三組小鼠間微膠細胞及星狀膠細胞數量差異不大，因此我們認為B6純品系3基因轉殖小鼠及B6.129雜交3基因轉殖小鼠沒有膠細胞過度增生的情況。我們在行為測試中發現B6.129雜交3基因轉殖小鼠發病情形較為嚴重，但在蛋白質含量測試上發現B6品系3基因轉殖小鼠tau磷酸化程度較高。因此，未來需要進一步確認B6品系3基因轉殖小鼠在阿茲海默氏症上研究的可行性。Alzheimer’s disease (AD) is the most common form of dementia and can be classified into two major types, the early-onset Familial Alzheimer’s disease (FAD) and the late-onset Sporadic Alzheimer’s disease (SAD). FAD is caused by genetic mutation, such as mutations of genes encoding amyloid precursor protein (APP), Presenilin-1 (PS1) and Presenilin-2 (PS2). SAD is caused by the environmental factors, including eating habits, stress, and age. AD pathological features are amyloid plaques formed by beta-amyloid (Aβ) accumulation outside of neurons and neurofibrillary tangle caused by tau protein hyperphosphorylation inside of neurons; these effects ultimately result in neuronal death. Current treatments can only slow down the course of AD, but cannot cure this disease. Animal model is critical for pathomechanism and therapeutic investigation. The 3TG mouse model, B6.129-Psen1tm1Mpm Tg (APPSwe, tauP301L) 1Lfa/J is a commonly used model for AD. However, this model only showed mild phenotype in our previous study and it is hard to prepare hybrid wild-type mice for comparison. We backcrossed the 3TG mice into pure B6 genetic background. We compared the locomotor activity, anxiety, short-term and long-term memory of these mice. We found that B6.129 3TG mice had lower locomotor activity and anxiety than B6 WT and B6 3TG mice. The short term memory analyzed by Y maze test showed B6 3TG mice and B6.129 3TG mice both have lower alternative rate at 12 month than B6 WT mice, and there is no difference between B6 3TG mice and B6 WT mice. In addition, we also analyzed the expression levels of many protein in hippocampus. We found that B6.129 3TG mice have higher Aβ level than WT mice at 12 month old, and B6 3TG mice have higher Aβlevel than WT mice at 18 month old. B6 3TG mice have higher tau and phosphorylation level at serine 202 and 396 at 12 and 18 month old than WT mice. The results of immunostaining of brain sections show that pS202Tau of B6 and B6.129 3TG mice is higher than WT mice since 9 months old. Among the several kinases that phosphorylate tau, we found that pERK level in B6 3TG mice is higher than WT and B6.129 3TG mice. In addition, we found that the pERK signal was colocalized with NeuN signal. Thus, we suggest that pERK might be the kinase involved in tau protein phosphorylation in B6 3TG mice. The Iba1 and GFAP staining in brain sections only have slight differences among three groups, which reveals B6 and B6.129 3TG models didn’t have the problem of gliosis. We found that B6.129 3TG mice have faster progression of disease, however, B6 3TG mice have higher tau phosphorylation. Therefore, we have to further investigate the feasibility of B6 3TG mice in AD study.阿茲海默氏症3基因轉殖鼠雜交品系遺傳背景AβTauAlzheimer’s disease3TG transgenic micehybrid straingenetic backgroundAβtau